This research will be done primarily in India at Bose Institute in collaboration with Anuradha Lohia as an extension of NIH grant # R01CA60499. [unreadable] [unreadable] In the previous cycle of the parent grant replication initiation factors in mammalian cells were identified. In the current cycle of the parent grant the aims are directed at understanding how these initiation factors are regulated through the cell-cycle with a particular emphasis on the role of checkpoint pathways in preventing re-replication of the genome. The FIRCA application will take advantage of a protist, E. histolytica, that undergoes repeated rounds of re-replication in the normal life cycle. In oreder to understand how the normal controls against re-replication are bypassed, the first aim will study the cell-cycle regulation of the MCM proteins in E. histolytica and correlate the same with replication and re-replication. This aim will test whether the MCM proteins are as important in E. histolytica, as they are in mammals, for replication initiation and whether inhibition of MCM proteins during S phase is critical for prevention of re-replication. Cyclin dependent kinases (cdk) are a major regulator of the mammalian cell cycle and play a critical role in preventing re-replication. In the second aim the role of individual cdks from E. histolytica in stimulating replication and preventing re-replication will be studied. If a cdk is found to be critical for prevention of re-replication, we will study whether that cdk phosphorylates ameba MCM proteins as they do in mammalian cells. [unreadable] [unreadable]